Ca
2 +
-independent PEPC kinase PEPCk is up- regulated via a light-dependent transduction cas-
cade [3]. In seeds, a heterotrophic PEPC whose activity is increased during maturation is thought
to provide carbon skeletons for the synthesis of amino and fatty acids [4 – 8], thus contributing to
accumulation of protein and lipid, while its role during germination is still unclear [9]. Consistent
with this view are recent results on barley seeds showing that the carbon flux through PEPC rel-
ative to pyruvate kinase increases 3 – 5-fold to- gether with a 5-fold increase in PEPC activity
during
maturation and
acidification of
the starchy endosperm which is due to
L
-malate ac- cumulation
[8]. Two
immunologically-related PEPC polypeptides 103 and 108 kDa have
been identified in a variety of seeds from C
3
plants like castor oil, wheat and barley [4,6,7]. Both polypeptides have been shown to be phos-
phorylated in vitro and in vivo during germina- tion of wheat seeds [6]. Recent results have
documented the fact that a Ca
2 +
-independent PEPCk is already present in dry barley seeds
and does not necessitate the functioning of a transduction cascade for up-regulation during
subsequent germination [9].
Very little is known about seed PEPC from graminaceous C
4
plants. It was reported that the enzyme and corresponding mRNA are synthe-
sized in imbibing Sorghum seeds [10]. In the present work, the fate and properties of Sorghum
seed PEPC have been investigated, paying partic- ular attention to the metabolite and salt effect
on the phosphorylation process of the enzyme during germination.
2. Materials and methods
2
.
1
. Plant material Sorghum 6ulgare var. Tamaran seeds were
sterilized in 5 vv NaOCl for 15 min and thoroughly washed with sterile water. Seeds were
germinated either on filter paper soaked in sterile distilled water or on sterile distilled water con-
taining different concentrations 50, 100, 150, 200 mM of NaCl, or in 10 mM Hepes – KOH,
pH 7 + 10 mM
L
-malate, in Petri dishes. The dishes were placed in darkness in a sterile cham-
ber at 24°C.
2
.
2
. Seed extracts Dry seeds were de-embryonated, or germinated
seeds separated from the seedling using a razor blade prior to extraction of soluble proteins. The
material ten seeds was chopped and ground thor- oughly on ice in a prechilled mortar with washed
sand in 1 ml of medium A containing 100 mM Tris – HCl, pH 8, 10 mM MgCl
2
, 1 mM EDTA, 5 vv glycerol, 14 mM b-mercaptoethanol, 10
mgml chymostatin, 10 mgml leupeptin, 1 mM PMSF. The homogenate was centrifuged at
120 000 × g for 5 min Beckman ultracentrifuge TL100, and the supernatant fluid was used as a
crude extract or rapidly filtered through Sephadex G-25 equilibrated with medium A without b-mer-
captoethanol desalted extract.
2
.
3
. Immunocharacterization of seed PEPC Proteins from desalted extracts were subjected
to 10 SDS-PAGE [11] for 2 h, 10 Vcm, at room temperature in a Bio-Rad electrophoresis cell.
Proteins were electroblotted overnight to a nitro- cellulose membrane at 30 V, 4°C in a Bio-Rad
transfer blot apparatus. The membrane was blocked at room temperature by a 4 h incubation
in TBS 20 mM Tris – HCl, 0.15 M NaCl, pH 7.9 containing 5 wv powdered milk. PEPC was
immuno-labeled by a 3 h incubation of the mem- brane at room temperature in 10 ml of TBS con
taining 20 mg of affinity-purified N-terminal IgG APS-IgG [12]. Subsequent detection was by a
peroxidase assay affinity-purified goat anti-rabbit IgG horseradish conjugate from Sigma.
2
.
4
. Purification of seed PEPCk This was performed as described by Ref. [13].
All steps were carried out at 4°C. De-embryonated dry seeds [20] were homogenized using a mortar
and pestle in 2 ml of medium A, and the brei ultracentrifuged at 120 000 × g as indicated above.
Ammonium sulfate was added to 60 saturation and precipitated proteins collected by centrifuga-
tion in an Eppendorf centrifuge. The protein pellet was disolved in 200 ml medium B containing: 50
mM Tris – HCl pH 7.8, 20 glycerol and 1 mM DTT and dialyzed against the same medium 2 ×
500 ml + 1 l for 2 h. Proteins were subjected to affinity chromatography on blue-dextran-agarose
BDA: 1 ml equilibrated in medium B at a flow rate of 0.05 mlmin. After thorough washing with
medium B, bound proteins were eluted with medium B containing 500 mM NaCl. Peak frac-
tions were pooled and precipitated with 60 satu- ration of ammonium sulfate, after which proteins
sedimented by centrifugation were resuspended in 100 ml of 50 mM Tris – HCl pH 8, 30 glycerol, 1
mM dithiothreitol, and stored at − 20°C until use.
2
.
5
. In 6itro phosphorylation The reconstituted phosphorylation assay 50 ml
system contained: 50 mM Tris – HCl, pH 8, 5 mM MgCl
2
, 0.04 mM CaCl
2
, 20 glycerol, 1 mM DTT, 74 kBq [g-
32
P]ATP 37 GBqmmol, 0.25 mM
P1P5-diadenosine-5-pentaphosphate an
adenylate kinase inhibitor, 4 mM phosphocre- atine, 10 U of creatine phosphokinase compo-
nents of the ADP-scavenging system, and an aliquot of the desalted crude extract 30 mg of
protein. In assays where partially, BDA-purified seed protein kinase was tested, 0.2 U6 mg of C
4
PEPCs S8; wild type or S8D, Ser8 replaced by aspartate were added as phosphorylation and
control targets [14]. These Sorghum PEPC forms were produced as recombinant proteins in Es-
cherichia coli and subsequently purified from bac- terial extracts by immunoaffinity chromatography
[15]. In some assays, the PEPC target was incu- bated in the presence of 10 mg of APS-IgG for 10
min at 4°C prior to in vitro phosphorylation test. After 45 min incubation at 30°C, the phosphoryla-
tion reaction was halted by addition of 10 ml of SDS sample buffer 50 mM Tris – HCl, pH 8, 1
wv SDS, 10 vv 2-mercaptoethanol, 20 v v glycerol and 1 wv bromophenol blue and
heated for 2 min at 100°C. Denatured samples were analyzed by SDSPAGE 10 acrylamide
according to [11], and autoradiographed.
2
.
6
. In situ
32
P-labeling and immunoprecipitation of seed PEPC
De-embryonated seeds ten were allowed to imbibe in 200 ml of distilled water containing
74 × 10
5
Bq of [
32
P]phosphate specific radioactiv- ity 74 × 10
2
GBqmol for 48 h at room tempera- ture. The seeds were washed thoroughly 5 times
with distilled water to remove remaining labeled phosphate and proteins were extracted in 1 ml
medium A as described above. The homogenate was centrifuged at 15 000 × g for 5 min. An
aliquot of the clarified sample containing 14.4 mU of PEPC was incubated overnight, 4°C, with the
appropriate amount of protein A-purified APS- IgG 20 mg of protein. Protein A Sepharose beads
were added to the incubated sample to 6 wv and vortexed briefly. The beaded immunocom-
plexes were washed five times with washing medium 500 mM Tris – HCl pH 8, 1.5 M NaCl
and 1 vv Triton X-100. The final pellet of centrifugation was solubilized in 100 ml of SDS
sample buffer, boiled for 5 min, and protein were separated by 10 SDS-PAGE [11] for 2 h at 100 V
and room temperature. Proteins were electrob- loted overnight to a nitrocellulose membrane at 30
V, 4°C in a Bio-Rad transfert blot apparatus. The membrane was dried and exposed to Kodak film
at − 80°C, then seed PEPC was immunocharac- terized as described in Section 2.3.
2
.
7
. PEPC acti6ity assays and
L
-malate sensiti6ity test
Unless otherwise stated, the standard assay con- ditions were as described in [2,16].
2
.
8
. Determination of proteins and
L
-malate Soluble protein concentration was measured ac-
cording to [17] using the Bio-Rad dye reagent and bovine serum albumin as a standard.
L
-malate concentration was determined in aliquots of the
seed crude extracts by a spectrophotometric assay in the presence of NAD-dependend MDH and
NADH according to [18].
3. Results
